The invention relates to a method for determining the number of sites of infection of a cell culture.
For determining the number of sites of infection of a cell culture, it is known to first grow a cell culture on a sample carrier. Suitable sample carriers are in particular titer plates and microtiter plates, wherein cell cultures are grown in the individual wells so that the cells can attach to the well bottom. Thereafter, the cells are infected with a viral liquid. The same is usually diluted, with viral liquids of different dilutions being added to different wells. After a predetermined period, which depends in particular on the type of virus, the viral liquid is removed, in particular pumped off. The viral liquid usually acts on the cell culture for several minutes to several hours. After removal of the viral liquid, a medium is usually supplied that is to prevent a diffusion of virus particles released at a later time. Such a medium, which is introduced in particular as a gel layer, is used to cover the cell culture. Thereafter, a longer period of typically several days of waiting occurs. During this period, the infected cells die and the viruses emerging therefrom spread into the adjacent cells. Thereby comparatively large areas are formed in which dead cells are no longer visible and are surrounded by zones with cells that are still alive but already infected. Here, sites of infection are defined as areas on the sample carrier in which an initial infection of the cells by the virus has taken place, wherein, starting from the respective site of infection, infected areas are formed in which the cells are already infected and/or have already been destroyed.
Using a transmitted light method, it is possible to count the destroyed areas or areas in which no living cells are left. Such counting is typically performed after taking a respective image by persons, by photography or microscopy. If necessary, the sample may be stained with a dye for an increase in contrast. A suitable, in particular unspecific dye, such as e.g. methylene blue, marks the living cells so that the areas, in which no living cells exist, are more clearly obvious to the viewer as holes.
Determining the number of sites of infection of a cell culture by means of a transmitted light method in particular has the disadvantage that e.g. cells infected at a later time or cells reacting to the virus in a different way have not died yet and, as such, no corresponding hole can be seen using the transmitted light method. This also applies e.g. to cells that react more slowly, so that the corresponding holes are still very small and are therefore not visible or only with difficulty. It is another disadvantage of the transmitted light method that other voids that were not caused by the viruses, are detected as such voids and are erroneously included in the count. Such voids may e.g. areas in which the cells were washed away or in which contaminations exist. Washing away can occur e.g. when the viral liquids are added or when the gel is added. Further, the duration of the period which has to be waited prior to the transmitted light method being performed correspondingly, is difficult to determine and depends in particular on the type of the viruses. If the period is too short, the corresponding infected areas only have a small number of dead cells so that the corresponding holes or voids are difficult to discern. If the period is too long, individual areas grow to merge so that it is hard to determine, whether, originally, there was only one site of infection, or a plurality of sites of infection with a common infected area of the cell culture.
Further, for determining the number of sites of infection of a cell culture, it is known to treat the cell culture with in particular virus-specific fluorescence makers that mark the infected cells. It is then possible to count the infected areas, since the corresponding areas fluoresce. However, this method has a disadvantage in that adjacent infected areas cannot be discerned from each other and can therefore only be detected erroneously as one original site of infection.